Pied-pipers wanted: The search for super-lures of New Zealand mammal pests

​Introduced mammalian competitors and predators are the leading threat to New Zealand’s native wildlife (Craig et al. 2000). The recent Pest Summit (3–4 December, 2012: Linklater 2013a) identified improving ways of detecting and killing mammals as the top three research priorities. One of these was the development of better lures to attract pest mammals to monitoring and killing devices. We have fought conservation battles largely with existing food-based lures. Mammals have been eradicated on islands and their populations depressed on the mainland. But we are not yet winning the war. Eradication of the worst mammal pests remains improbable on the mainland because current technolo-gies cannot operate at the required scale and intensity within probable budgets. The extensive deployment of killing devices for extermination at greater scales is juxtaposed logistically against the intensity of effort required to kill the few last, most difficult to detect, animals

An open-format enteroid culture system for interrogation of interactions between Toxoplasma gondii and the intestinal epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection

A novel <i>in vitro</i> model of the small intestinal epithelium in co-culture with ‘gut-like’ dendritic cells

Abstract Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated ‘gut-like’ DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/− (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These ‘gut-like’ DCs extended transepithelial dendrites across the intact epithelium of enteroids. ‘Gut-like’ DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in ‘gut-like’ DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.</jats:p

Studies of seasonality in red deer (Cervus elaphus) : with special emphasis on the reproductive physiology of red deer hinds

Four trials were conducted to investigate factors controlling the seasonal onset of reproductive activity in red deer hinds. Firstly (Chapter 4), the role of photorefractoriness to long daily photoperiods in the initiation of the seasonal reproductive activity in breeding red deer hinds was examined. Red deer hinds (n=10) were prematurely exposed to a long daily photoperiod of 15.3 h from 22 July to 8 November 1986 i.e. winter-spring (EPW), or maintained under natural photoperiods (NP). Six hinds experienced the natural changes in daily photoperiod until mid-summer but were exposed to a 15.5 h of light each day from 30 January to 30 April 1987, i.e. summer-autumn (EPS), whilst hinds in the other groups experienced naturally decreasing daily photoperiods. On 5 occasions (July 1986, January, February, March and April 1987), blood samples were collected from 4 NP and 4 EPW hinds every 20 minutes for 4 h to monitor secretion of luteinising hormone and half hourly for another 4 h following an i.v. injection of 2 µg GnRH to measure pituitary responsiveness. In January, March and April 1987 EPS hinds were also intensively sampled for 4 h. Plasma progesterone concentrations and mean date of calving indicated that the onset of breeding activity was not affected by light treatment in the EPW hinds but was delayed by 3 weeks in the EPS hinds. In contrast, supplementary lighting caused a premature elevation of plasma prolactin concentrations and advanced pelage moulting in EPW hinds only. Plasma LH secretion patterns indicated that LH pulse frequency and mean LH concentrations were greater during the breeding season than during pregnancy or, seasonal or postpartum, anoestrus. The reduction in LH secretion was partially explained by a diminished pituitary responsiveness to GnRH. Daily plasma melatonin secretion patterns indicated that the duration of the nocturnal increase in melatonin concentrations was responsive to changes in photoperiod and provided a suitable endocrine signal for measuring day length. The results suggest that, unlike the sheep and prepubertal red deer hind, the onset of seasonal breeding activity in breeding red deer hinds is not affected by long daily photoperiods in spring but is delayed if the autumnal decrease in daily photoperiod is delayed. Therefore neither the development of photorefractoriness nor the spring increase in daily photoperiods initiated the transition from seasonal anoestrus to reproductive activity in the breeding hind. However, long daily photoperiods may have entrained the annual cycle of pelage shedding and prolactin secretion. It is possible that the neuroendocrine pathway by which photoperiodic signals entrain the seasonal cycle of reproduction is separate from those which regulate other seasonal events in the breeding red deer hind. Secondly (Chapter 5), in a study of seasonality of reproduction, 4 pubertal hinds were monitored for live weight and plasma LH and progesterone concentrations from December 1987 to October 1988 (i.e. 12-22 months of age). In addition the pattern of LH secretion was also studied in 4 ovariectomised pubertal hinds implanted s.c. with controlled release implants containing 12 mg oestradiol-17β between 4 March and 25 May and between 15 June and 19 September, 1988. On several occasions (15 December, 29 February, 15 March, 24 April, 14 June, 29 June, 18 September (all hinds) and 3 October (ovariectomised hinds only) blood samples were collected every 20 minutes for 4 h to monitor secretion of luteinising hormone and following an i.v. injection of 2 µg GnRH to measure pituitary responsiveness. Plasma progesterone profiles indicated that 4-6 ovarian cycles, lasting about 19 d each, occurred in each intact hind. Regular ovarian cycles commenced in late April (26 April ± 3.4 d, mean ± s.e.m.) and ceased 3 months later in July (21 July ± 7.2 d). The number of LH pulses in the intact hinds was higher in June (1-2 pulses/4 h) than in the non-breeding season (< 1 pulse/4 h) probably due to a seasonal increase in GnRH secretion. It appears that the seasonal increase in LH pulsatility and onset of reproductive activity in the entire hinds were temporally related to a reduction in the sensitivity of LH secretion to the negative feedback effects of oestradiol in ovariectomised pubertal hinds. In the third trial (Chapter 6), 20 male and 20 female red deer calves were immunised at birth against a melatonin conjugate or injected with adjuvant only (controls). Booster injections were given on 5 occasions over the next 2 years. Stags which produced significant melatonin binding activity in response to immunisation, were heavier than the controls between 7 and 11 months of age and at 16, 20, 30 and 34 months of age. A similar but smaller effect on live weight was seen in the immunised hinds. Immunisation against melatonin did not affect the calving date of the hinds or antler development and time of casting of antlers in the stags. These results indicated that disruption of the photoperiodic signal by immunisation against melatonin may have prevented the entrainment of annual rhythms in feed intake and growth but without affecting the seasonal cycles in antler growth and ovarian activity. Finally in a series of 3 trials (Chapter 7), anoestrous red deer hinds were induced to ovulate with the GnRH analogue, buserelin. Hinds were pre-treated with intravaginal devices containing 0.6 g progesterone (CIDR-Type S) for 14 days prior to CIDR withdrawal on 4 March. In Year 1, 15 hinds were treated with 1 CIDR each and 8 hinds were injected i.m. with 4 µg buserelin (a GnRH analogue) at CIDR removal followed by 2nd injection of 10 µg 48 h later. In Year 2, 16 hinds were treated with 2 CIDRs each and 8 hinds were injected Lm. with 4, 3, 2, 2 and 10 µg buserelin at -48, -24, 0, 24 and 48 h respectively from CIDR withdrawal. In Year 3, 15 hinds were treated with 2 CIDRs and 11 hinds (Groups Band BO) injected with buserelin identical to the protocol in Year 2. At CIDR withdrawal 6 of these buserelin treated hinds (Group BO) and 4 other progesterone-primed hinds (Group 0) were injected with 500 µg oestradiol benzoate. In addition, during the breeding season, 4 hinds (Group C) were treated with double CIDRs only for 14 d from 1 April. When oestrous behaviour and the pattern of plasma LH secretion were monitored in Year 3, oestrous behaviour was less noticeable and delayed in Group B hinds and peak LH levels were lower and increased later relative to Group 0, BO and C hinds. Two weeks after CIDR withdrawal, 6 buserelin-treated hinds in both Year 1 and Year 2, and 3/5 B, 2/4 0, 3/6 BO and 4/4 C group hinds in Year 3 had a single corpus luteum present. Plasma progesterone concentrations were elevated for about 12 d in most hinds with a corpus luteum in Year 2 and in most B, BO and C Group hinds in Year 3. However, progesterone secretion was low in several Year 1 and all Group 0 hinds in Year 3 indicating that the induced corpora lutea in these hinds were functionally subnormal. There was no evidence that any of the buserelin-induced ovulations resulted in pregnancy, probably because the induced ovulations were not accompanied by normal hormonal and behavioural patterns. The experiments described in this thesis have contributed to our understanding of the regulation of seasonal breeding in red deer hinds. This knowledge may eventually enable reproductive activity to be effectively manipulated, thereby improving the efficiency of deer production systems

Research To Develop Contraceptive Control of Brushtail Possums in New Zealand

Common brushtail possums are serious pests in New Zealand, where they threaten the survival of native plants and animals and spread bovine tuberculosis. A National Science Strategy Committee established in 1991 to coordinate possum research gave high priority to research aimed at biological control of possums, particularly contraceptive control. Surveys are identifying pathogens and potential vectors, and research has begun on immunology, gene transcription, potential contraceptive targets, and sociobiology. As there are more than 60 million possums in New Zealand, contraceptive vaccine delivery systems need to be cost effective, and they must be publicly acceptable. A vaccine could be included in a bait, but long-term cost-effective control will probably require a biological vector. Eventually the best control strategy will probably combine traditional control and immunocontraception

MHC haplotypes and response to immunocontraceptive vaccines in the brushtail possum

The possum is a major invasive pest in New Zealand. One option for its control is the use of immunocontraceptive vaccines. Initial trials of vaccines have shown individual variation in response. The use of vaccines on wild populations could result in the evolution of a resistant population through selection for possums that remain fertile because of low or no response. Understanding the basis of this variation is therefore important. The major histocompatibility complex (MHC) is an important influence on the nature of immune responses. This study has investigated the relationship between MHC alleles and individual immune responses to immunocontraceptive vaccines comprising zona pellucida peptides. We identified MHC alleles and putative haplotypes, and compared these between individuals with measured responses to immunocontraceptive vaccines. Two haplotypes were found to associate significantly with differences in vaccine response. Possums that carried haplotype 6 showed reduced responsiveness to one vaccine, while possums that carried haplotype 9 showed increased responsiveness to a separate vaccine. The identification of MHC haplotypes associated with different responses to immunocontraceptive vaccines offers the opportunity to understand what factors trigger non-response and the persistence of fertility in some individuals, and may allow vaccines to be optimised to minimise non-responsiveness.</p

Fertility Control for Wildlife Management The Brushtail Possum in New Zealand

Fertility control vaccines are under development for a number of pest wildlife species, mostly based on whole zona pellucida (ZP) or individual ZP proteins. Such vaccines must be effective, long lasting, cheap and readily deployed. One approach to deployment is oral delivery in baits. This is one strategy being taken in New Zealand for control of brushtail possums, 2- to 4-kg marsupials introduced from Australia in the 1850s, and now major pests of both conservation and agricultural production. New Zealand has highly effective aerial and ground systems for delivery of toxic baits to possums that could be adapted readily to deliver vaccine baits. Recent trials in captivity where female possums were immunised with recombinant possum ZP3 and ZP2 proteins demonstrated 70-75% reductions in fertility in natural and assisted breeding trials. Immunisation with possum-specific epitopes of the ZP2 and ZP3 proteins has also proved effective at reducing the numbers of fertilised eggs recovered from immunised females. For field delivery of an oral vaccine, we are investigating the use of bacterial “ghosts”. These are the empty cell walls of bacteria that have been modified to express possum ZP proteins in their cell walls. The possum’s immune system recognises the bacterial ghost as foreign, and produces antibodies against them. At the same time, it is tricked into developing antibodies against the possum egg proteins, causing a contraceptive effect. In a recent proof of concept trial, female possums immunised with a possum ZP2-bacterial ghost vaccine by nasal spray showed a significant reduction in fertility. Oral delivery may require protection of vaccine ghosts from degradation. We have developed a protective system and are currently repeating this trial using oral delivery of the ZP2 ghost vaccine. Our future priority is increasing the vaccine efficacy and longevity ahead of limited field trials in 2009

Fertility Control for Wildlife Management The Brushtail Possum in New Zealand

Fertility control vaccines are under development for a number of pest wildlife species, mostly based on whole zona pellucida (ZP) or individual ZP proteins. Such vaccines must be effective, long lasting, cheap and readily deployed. One approach to deployment is oral delivery in baits. This is one strategy being taken in New Zealand for control of brushtail possums, 2- to 4-kg marsupials introduced from Australia in the 1850s, and now major pests of both conservation and agricultural production. New Zealand has highly effective aerial and ground systems for delivery of toxic baits to possums that could be adapted readily to deliver vaccine baits. Recent trials in captivity where female possums were immunised with recombinant possum ZP3 and ZP2 proteins demonstrated 70-75% reductions in fertility in natural and assisted breeding trials. Immunisation with possum-specific epitopes of the ZP2 and ZP3 proteins has also proved effective at reducing the numbers of fertilised eggs recovered from immunised females. For field delivery of an oral vaccine, we are investigating the use of bacterial “ghosts”. These are the empty cell walls of bacteria that have been modified to express possum ZP proteins in their cell walls. The possum’s immune system recognises the bacterial ghost as foreign, and produces antibodies against them. At the same time, it is tricked into developing antibodies against the possum egg proteins, causing a contraceptive effect. In a recent proof of concept trial, female possums immunised with a possum ZP2-bacterial ghost vaccine by nasal spray showed a significant reduction in fertility. Oral delivery may require protection of vaccine ghosts from degradation. We have developed a protective system and are currently repeating this trial using oral delivery of the ZP2 ghost vaccine. Our future priority is increasing the vaccine efficacy and longevity ahead of limited field trials in 2009

Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs